This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Phytochromes (Phys) are a diverse collection of photoreceptors that regulate an array of important processes in plants and bacteria by binding heme-derived linear tetrapyrroles to become photochromic. The canonical Phys consist of two spectral states, a red-absorbing ground state (Pr) and a far red-absorbing excited state (Pfr). Recent genomic and biochemical analyses have revealed a divergent subset of Phy-like blue-green photochromic proteins unique to cyanobacteria, the cyanobacteriochromes (Cbcs). Unlike canonical red-absorbing cyanobacterial Phys, which rely on a single cysteine for attachment of the phycocyanobilin (PCB) chromophore, Cbcs rely on a second conserved cysteine that enables their hallmark blue-green (Pb-Pg) photoreversible properties, presumably by shortening the p conjugation system of PCB. Using the photochromic GAF domain chromopeptide from Thermosynechococcus elongatus BP-1 protein tll0569 (TePixJ-GAF) as a model Cbc, we demonstrated by mutagenesis that the second Cys (Cys-494) indeed forms a covalent linkage to the bilin. Absorbance, IR, NMR, and RR spectroscopic techniques show that the additional Cys494-PCB adduct most likely occurs at the tetrapyrrole A-B methine bridge, yielding a more de-conjugated phycoviolobilin-like tetrapyrrole. This linkage remains intact throughout the photocycle, with all four PCB nitrogens being protonated in both Pb and Pg states. NMR spectroscopy with uniformly isotopically-labeled 15N-TePixJ-GAF polypeptide revealed that Pb and Pg states may be maintained as two entirely distinct structures without evidence of dark reversion nor cross contamination. We will attempt structure determination of the TePixJ-GAF domain which houses the chromophore. The construct photoconverts and is only ~20 kDa;we would like to also determine its Pb andf Pg forms upon blue/green light exposure.